Comparison of Mouse Ly5a and Ly5b Leucocyte Common Antigen Alleles

Journal of Immunology Research, Aug 2018

The family of leucocyte common antigen (LCA) transmembrane glycoproteins is expressed in most hematopoietic cells. Molecular isoforms of the LCA molecule are generated by alternative splicing of a single gene encoded on the murine chromosome 1. Three LCA alleles with different antigenic reactivities have been identified in inbred mouse strains. To investigate the divergence between alleles, cDNA clones to the SJA (Ly5a) LCA gene have been isolated and sequenced. A comparison of this information to the Ly5b allele sequence identifies 12 allele-specific nucleotide changes. These base substitutions correspond to five amino-acid changes within the extracellular domain of the LCA molecule. These amino-acid differences are clustered in a region that also contains the greatest divergence between mouse and rat LCA sequences. Thus, these two mouse LCA alleles exhibit a pattern of sequence conservation that mimics that found over a much broader scale of evolution. Analysis of antigenicity profiles for each of the allelic sequence changes reveals three molecular domains of altered antigenicity that could account for observed serological differences between the two alleles. Sequence information from the 5

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Comparison of Mouse Ly5a and Ly5b Leucocyte Common Antigen Alleles

Comparison of Mouse Ly5a. and Ly5b Leucocyte Common Antigen Alleles SUZANNE L. ZEBEDEE 0 DIANA S. BARRITT 0 WILLIAM C. RASCHKE 0 0 La Jolla Biological Laboratories, The Salk Institute , P.O. Box 85350, San Diego, California 92186 , USA The family of leucocyte common antigen (LCA) transmembrane glycoproteins is expressed in most hematopoietic cells. Molecular isoforms of the LCA molecule are generated by alternative splicing of a single gene encoded on the murine chromosome 1. Three LCA alleles with different antigenic reactivities have been identified in inbred mouse strains. To investigate the divergence between alleles, cDNA clones to the SJA (Ly5a) LCA gene have been isolated and sequenced..A comparison of this information to the Ly5b allele sequence identifies 12 allele-specific nucleotide changes. These base substitutions correspond to five amino-acid changes within the extracellular domain of the LCA molecule. These amino-acid differences are clustered in a region that also contains the greatest divergence between mouse and rat LCA sequences. Thus, these two mouse LCA alleles exhibit a pattern of sequence conservation that mimics that found over a much broader scale of evolution. Analysis of antigenicity profiles for each of the allelic sequence changes reveals three molecular domains of altered antigenicity that could account for observed serological differences between the two alleles. Sequence information from the 5' end of the Ly5 LCA gene, generated using polymerase chain-reaction techniques on genomic DNA, reveals eight additional nucleotide differences between the Ly5 and Ly5b alleles. cDNA; domain; epitopes; evolution INTRODUCTION The leucocyte common antigen family of glycoproteins is abundantly expressed on the surface of most cells in .the hematopoietic lineage (Scheid and Triglia, 1979; Sarmiento et al., 1982) . These antigens (also known as CD45, Ly5, T200, and B220) have been identified in mouse, rat, chicken, and human systems (Komuro et al., 1975; Fabre and Williams, 1977; Judd et al., 1980; Omary et al., 1980; Houssaint et al., 1987) . Data from the analysis of cDNA clones indicate that the murine LCA molecule has a hydrophobic leader sequence, an N-terminal extracellular domain consisting of 402-.541 amino acids, a single transmembrane region, and a large cytoplasmic domain of 705 residues (Saga et al., 1986, with a correction, 1987; Thomas et al., 1987) . The mapping of this family as a single gene on the mouse chromosome 1 has been completed and shown to *Corresponding author. comprise 34 exons (Saga et al., 1988) ; exons la and l b are alternatively excluded 5? untranslated sequences of LCA mRNAs, and exons 2-33 are protein-encoding. The function of the leucocyte common antigen has not been determined, although its involvement in leucocyte activities, such as natural killer cytolysis (Sparrow and McKenzie, 1983), cytotoxic T-cell cytolysis (Harp et al., 1984) , as well as lymphocyte activation and differentiation (Yakura et al., 1986; Mittler et al., 1987; Pingel and Thomas, 1989) has been implicated. These roles may be mediated through the tyrosine phosphatase activity shown to be associated with the LCA molecule (Tonks et al., 1988) . In the mouse, the LCA molecule shows heterogeneity in molecular weight, glycosylation, and antigenicity patterns. Differences in LCA family members have been traced to the alternative splicing of exons 4, 5, and 6, which generates multiple molecular isoforms (Saga et al., 1987; Thomas et al., 1987) . In B lymphoid cells, the major LCA protein product is 220,000 m.w. and contains all three of these alternative exons, whereas the major thymocyte protein is 180,000 m.w. and does not include exons 4, 5, or 6. These differences account for an insertion of 139 residues in the extracellular domain of the B-cell expressed LCA molecule. Proteins of intermediate size have been observed, which apparently represent splice combinations of exons 4, 5, and 6 (Chang et al., 1989) . Glycosylation differences in the LCA protein family are also due to the inclusion of these variable exon sequences as the inserted amino acids contain many serine and threonine residues potentially linked to O-type carbohydrate structures (Childs et al., 1983; Johnson et al., 1989) . Murine LCA glycoproteins bear the Ly5 alloantigenic determinant, which has been characterized in both inbred and wild strains of mice (Seldin et al., 1987) . Inbred mice have been categorized into three LCA alleles, Ly5a, Ly5b, and Ly5c, which can be distinguished antigenically by their reactivity with specific monoclonal and alloantibodies (Ly5.1, Ly5.2) and genetically by RFLPs of mouse genomic DNA (Seldin et al., 1987) . Most established murine strains express the Ly5b allele, including BALB/c, C57BL, CBA/J, and NZB/BLNJ, yet SJL/J mice carry the Ly5 allele. To examine the differences between the murine Ly5 and Ly5b alleles, we have isolated and analyzed cDNA clones from t (...truncated)


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Suzanne L. Zebedee, Diana S. Barritt, William C. Raschke. Comparison of Mouse Ly5a and Ly5b Leucocyte Common Antigen Alleles, Journal of Immunology Research, 1, DOI: 10.1155/1991/52686